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com/esps/ World J Stem Cells 2016 October 26; 8(10): 316-331
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DOI: 10.4252/wjsc.v8.i10.316
Update on acute myeloid leukemia stem cells: New
discoveries and therapeutic opportunities
Maximilian Stahl, Tae Kon Kim, Amer M Zeidan
Maximilian Stahl, Tae Kon Kim, Amer M Zeidan, Section of
Hematology, Department of Internal Medicine, Yale School of
Medicine, New Haven, CT 06510-3222, United States
Author contributions: Stahl M and Zeidan AM wrote the first
draft of the manuscript; Stahl M, Kim TK and Zeidan AM edited
the manuscript.
Conflict-of-interest statement: Amer M Zeidan receives
Honoraria from Ariad, Pfizer, Incyte and Celgene. Maximilian
Stahl and Tae Kon Kim have no conflict of interests to declare for
this article.
Open-Access: This article is an open-access article which was
selected by an in-house editor and fully peer-reviewed by external
reviewers. It is distributed in accordance with the Creative
Commons Attribution Non Commercial (CC BY-NC 4.0) license,
which permits others to distribute, remix, adapt, build upon this
work non-commercially, and license their derivative works on
different terms, provided the original work is properly cited and
the use is non-commercial. See: http://creativecommons.org/
licenses/by-nc/4.0/
Manuscript source: Invited manuscript
Correspondence to: Amer M Zeidan, MBBS, Assistant
Professor, Section of Hematology, Department of Internal
Medicine, Yale School of Medicine, 300 George Street, New
Haven, CT 06510-3222, United States. amer.zeidan@yale.edu
Telephone: +1-203-2004363
Fax: +1-203-2002360
Received: May 23, 2016
Peer-review started: May 23, 2016
First decision: July 6, 2016
Revised: August 11, 2016
Accepted: August 27, 2016
Article in press: August 29, 2016
Published online: October 26, 2016
Abstract
The existence of cancer stem cells has been well
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ISSN 1948-0210 (online)
© 2016 Baishideng Publishing Group Inc. All rights reserved.
REVIEW
established in acute myeloid leukemia. Initial proof
of the existence of leukemia stem cells (LSCs) was
accomplished by functional studies in xenograft models
making use of the key features shared with normal
hematopoietic stem cells (HSCs) such as the capacity
of self-renewal and the ability to initiate and sustain
growth of progenitors in vivo . Significant progress
has also been made in identifying the phenotype and
signaling pathways specific for LSCs. Therapeutically, a
multitude of drugs targeting LSCs are in different phases
of preclinical and clinical development. This review
focuses on recent discoveries which have advanced
our understanding of LSC biology and provided rational
targets for development of novel therapeutic agents.
One of the major challenges is how to target the self-
renewal pathways of LSCs without affecting normal
HSCs significantly therefore providing an acceptable
therapeutic window. Important issues pertinent to the
successful design and conduct of clinical trials evaluating
drugs targeting LSCs will be discussed as well.
Key words: Leukemia stem cells; Cancer stem cells;
Acute myeloid leukemia; Stem cell niche; Xenotrans­
plantation; Plerixafor; NF-kB; C-X-C chemokine receptor
type 4
© The Author(s) 2016. Published by Baishideng Publishing
Group Inc. All rights reserved.
Core tip: Leukemia stem cell (LSC) directed therapy
targets: (1) Cell surface markers expressed on LSC:
CD33, CD44, CD123, CD47, etc. ; (2) Crucial pathways
for maintenance of their stemness: NF-κB, PI3K/
AKT/mTOR and bcl-2; and (3) Interactions between
LSC in the bone marrow niche: LSC mobilization with
granulocyte-colony stimulating factor and inhibition of
LSC homing to the bone marrow by interrupting the
C-X-C chemokine receptor type 4-CXCL12 and VCAM-
VLA4 axis.
316 October 26, 2016|Volume 8|Issue 10|

Stahl M, Kim TK, Zeidan AM. Update on acute myeloid
leukemia stem cells: New discoveries and therapeutic opportuni­
ties. World J Stem Cells 2016; 8(10): 316-331 Available from:
URL: http://www.wjgnet.com/1948-0210/full/v8/i10/316.htm
DOI: http://dx.doi.org/10.4252/wjsc.v8.i10.316
INTRODUCTION
Despite extensive research efforts in myeloid malig­
nancies, minimal progress has been made in intro­
ducing new effective treatment strategies for acute
myeloid leukemia (AML) since the introduction of the
anthracycline-cytarabine combination chemotherapy
regimens (known as 7 + 3) more than 40 years
[1]
ago . Despite achieving complete remission (CR) with
intensive induction chemotherapy in about 70% of
patients with AML, relapse is frequent and the rate of
5-year disease free survival is only about 30%-40%. It
has been long proposed that the high rate of relapse is
due to the persistence of a rare subset of malignant cells
that are not effectively eliminated by current treatment
[2-4]
regimens, the so called leukemia stem cells (LSCs) .
LSC were first identified but tumor cells with stem
cell-like behavior were later found to be also present
[5-9]
in a variety of solid tumors . LSC remain the best
studied and characterized cancer stem cell (CSC) due
to the easy accessibility of tumor tissue for (i.e., blood
and bone marrow) and the availability of a number
of cell surface markers that allow their prospective
identification and isolation by flow cytometry followed
by assays to examine their function both in vitro and in
[10]
vivo . This review will focus on the biology of LSC, the
impact they have on current leukemia diagnosis and
prognosis and treatment as well as future directions of
[6]
leukemia therapy based on targeting LSC .
CSC VS CLONAL EVOLUTION THEORY
It is now well understood that not only tumors from
different patients but also cells within a single tumor
are characterized by heterogeneity in terms of the
morphology, cell surface markers, genetic variations
[11]
and response to therapy . Why there is significant
variation in genetic and epigenetic abnormalities
between different cells or locations within a tumor
despite the clonal origin of all tumor cells, is a question
that has puzzled researchers for decades. There
are essentially two different explanations for this
fundamental problem of cancer biology: The hierarchy
or CSC model vs the stochastic or clonal evolution
[6]
model . In the stochastic model, all cells in a tumor
have a similar biological function but are heterogeneous
(e.g., expression of cell surface markers) because of
clonal evolution resulting in small but entirely random/
stochastic variations triggered by external and internal
factors based on Darwinian principles. Importantly, all
cells within the tumor have an equal sensitivity to both
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Stahl M et al . AML stem cells
intrinsic (transcription factors and signaling pathways)
and extrinsic (host factors, tumor microenvironment
[10]
and immune response) factors . In the cancer stem
cell (CSC) model, a tumor follows the prin­ciples of
normal, healthy tissue development with a stem cell
at the top of the hierarchy, which gives rise to all
other cells in the tumor. In this model only these rare
population of CSCs are able to initiate tumor growth:
They possess self-renewal capacity and can be isolated
from the bulk non-tumorigenic population. Importantly,
both models appreciate the existence of a CSC but
differ in their assessment what cells within the tumor
can be CSCs. In the stochastic model CSCs are created
randomly and every cell has the potential to be a CSC,
whereas in the CSC model only a subset of cancer cells
[11]
has the potential to behave like a stem cell .
Whether the stochastic model or the CSC model best
reflects tumorigenesis/leukemogenesis, has significant
impact on how cancer/leukemia should be treated .
[10]
In the stochastic model, the cells within a tumor are
relatively homogeneous in terms of genetic makeup
and function and therapy can be uniformly directed at
the bulk of tumor cells. However, per the CSC model,
tumorigenic pathways might operate differently in
CSCs compared with the bulk cells and therapy must
specifically target the CSCs in order to be truly effective.
Most of the current targeted therapies against leukemia
and cancer focuses on inhibiting the molecular drivers
found in all cancer cells but do not necessarily target
CSCs .
[11]
BIOLOGY OF LSCS
CSC characteristics
The definition of a LSC is adapted from normal HSC:
It is a cell that possesses the capacity to self-renew, pro­
liferates and gives rise to leukemic blasts, which are
morphologically homogeneous but biologically hetero­
[12]
geneous . Apart from self-renewal potential, dor­
mancy/quiescence and a protective stem cell niche are
shared characteristics between HSCs and LSCs.
Self-renewal capacity: As the definition of CSCs is
a functional definition, CSCs can thus only be defined
experimentally by their ability to recapitulate the
generation of a continuously growing tumor. Immuno­
deficient mice, such as the non-obese diabetic/severe
combined immunodeficiency (NOD/SCID) mouse
and newer generations of xenograft models, are used
to functionally define human hematopoietic stem
[13]
and progenitor cells as well as LSCs . Long-term
repopulating cells, thought to be LSC are able to be
successfully engrafted in these mice over prolonged
periods as well as in secondary recipients
[2,14]
. Bonnet et
[15]
al in the John Dick laboratory isolated subpopulations
of cells from primary human AML bone marrow based
on their immunophenotype and xenotransplanted
them into NOD/SCID mice. It demonstrated that the
+ -
CD34 CD38 expressing sub-population of AML cells
317 October 26, 2016|Volume 8|Issue 10|
 Stahl M et al . AML stem cells

were capable of being serially transplanted in these limited amount of cell divisions and regenerate all cells in
[15,16] [33,34]
immunodeficient mice . Reflecting the emphasis on a tissue .
functional assessment, these cells were named as SCID Similarly, LSCs are quiescent, which explains the
leukemia-initiating cells (SL-IC) and are considered the difficulties to eradicate LSCs with standard chemo­
equivalent of LSC. therapies that preferentially target rapid proli­ferating
[35-37]
cells .
Symmetrical vs asymmetrical cell division: Similar
to HSCs, LSCs have the ability to undergo symmetrical Key signaling pathways relevant for retaining
self-renewing cell division, generating identical daughter stemness: Similar signaling pathways involved in the
stem cells that retain self-renewal capacity (expansion), control of self-renewal of HSCs are also key elements
or an asymmetrical self-renewing cell division, resulting maintaining stemness in LSCs (Figure 1). Among many
[38]
in one stem cell and one more differentiated progenitor others, these pathways include PI3K/Akt/mTOR ,
[12,17-19] [39,40] [41,42] [43,44]
cell (maintenance) . Normal stem cells are able Wnt/beta-catenin , Hedgehog , NF-kB ,
[45] [46,47]
to switch between symmetrical and asymmetrical Notch and Bcl-2 . Several drugs targeting these
division based on the demands of the tissue they are pathways are in different stages of preclinical and
meant to maintain. During early embryogenesis normal clinical development (Figure 1).
stem cells undergo symmetrical cell division in order to
expand the total pool of stem cells giving rise to tissues Stem cell niche: The bone marrow niche is quintes­
whereas in adult tissues stem cells give rise to mature sential for normal HSC to maintain their quiescence
[19,20]
cells though asymmetrical cell division . There is but at the same time enable HSC to generate cells in
[48]
increasing amount of evidence that in CSCs this delicate the blood stream to meet the organism’s needs .
balance seems to be disturbed in favor of symmetric cell The stem cell niche is formed by a complex network
[19,21,22]
division . For example, CSCs isolated from ERBB2- of different cells including vascular endothelial cells,
expressing breast cancer have been demonstrated to perivascular mesenchymal cells, megakaryocytes,
prefer symmetric cell division compared to normal breast osteoblastic lineage cells, macrophages and nerve
[23] [49-53]
tissue stem cells . Furthermore, the adenomatous cells . Dysregulation of the bone marrow niche plays
polyposis coli tumor suppressor gene (APC) has been an important role in preventing the detection of LSC
shown to paly a major role in regulating asymmetric cell by the immune system and pro­tecting LSC from the
[48,54]
division in drosophila and its mutational loss is suspected effects of chemotherapy . Similar to normal HSCs,
to lead to an expansion of CSCs by symmetric cell LSCs are retained in the marrow niche by interactions
[22,24,25]
division . between CXCR4, on stem cells, and CXCL12 (SDF-1), on
osteoblasts and mesenchymal cells in the bone marrow
[55,56]
Stem cell quiescence and exhaustion: Normal stem niche . Chemokine interactions through CXCL12
cells need to be quiescent to avoid exhaustion of a stem can lead to up-regulation of vascular cell adhesion
[26]
cell pool and to minimize the risk of oncogenic events . molecule-1 (VCAM-1) and very late antigen-4 (VLA-4)
In fact, stem cell exhaustion has been described as one expression, which further strengthen LSC retention in
[57,58]
reason for aging and as a consequence of the attempt the marrow niche (Figure 1). The significance of
[27]
of the body to prevent the development of cancer . the interaction between LSCs and the protective bone
Aging leads to an accumulation of DNA damage in all marrow niche is exemplified by the fact that elevated
cells of the body, including stem cells, which in turn levels of CXCR4 and VLA-4 have been associated
leads to an increased risk of developing cancer. Aging with poor response to chemotherapy and decreased
[59-61]
stem cells are affected by sophisticated mechanisms survival . Several therapeutic approaches attempt
cells have developed to suppress the development of to break the dormancy of LSCs by induction of stem
cancer, mainly induction of senescence and apoptosis, cell cycling with granulocyte-colony stimulating factor
which are mediated through telomere shortening and (G-CSF) and inhibition of the CXCR4-SDF-1 axis
[28-30]
activation of tumor suppressor genes p16 and p53 . involved in LSC retention in the protective bone marrow
[62,63]
The diminished ability of aging HSC to reconstitute the niche (Figure 1).
hematopoietic system is demonstrated by prolonged
myelosuppression after cytotoxic chemotherapy in older Identification of LSCs by surface markers
patients as well as age of the stem cell donor being Recent studies have shown that LSCs may reside not
+ - + + -
significantly associated with overall and disease-free only in CD34 CD38 , but also in CD34 CD38 and CD34
[31,32] +
survival after hematopoietic stem cell transplant . CD38 compartments demonstrating the lack of a
[64-66]
However, normal stem cells are also required to definitive phenotype for LSCs . Several studies have
+ +
continuously replenish the cells that are lost in a tissue. shown that the CD34 CD38 fraction has repopulating
[18,67,68]
In order to fulfill both purposes-avoid exhaustion as well ability when immunosuppression is applied . It
as maintaining the cellular integrity of a tissue-stem cells was demonstrated that by treating mice with immuno­
+ +
undergo asymmetric cell divisions, which give rise to suppressive antibodies, the CD34 CD38 fraction of
another stem cell as well as a rapidly dividing progenitor AML samples is able to initiate leukemia in immuno­
[64]
cells. These progenitor cells proliferate quickly for a deficient mice . Furthermore, by transplanting sorted

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 Stahl M et al . AML stem cells

H90
Gemtuzumab ozogamicin
A3D8
Hu5F9-G4 (mylotarg)
CSL360
DT388IL3/SL-401
MGD006/S80880

Natalizumab

CD44
CD33

Bortezomib CD47 CD123

VLA-4 Stromal
Pr
ot cell
ea VCAM-1
so
m
e CXCR-4 Homing of LSC in
SDF
the bone marrow
niche
Bcl-2
IkBa

t
las
Plerixafor

ob
NF-kB AMD3465

te
Os
mTOR BMS-936564
GC
Parthenolide SF
PI3K Oblimersen -R
celastrol
Obatoclax
4-hydroxy- Br G-CSF
AKT ing
2-nonenal ing
LS
BKM120 C G-CSF
in
CAL-101 cy
cle
GSK21110183 Sirolimus,
MK-2206 everolimus,
Therapy targeting LSC surface markers
Perifosine temsirolimus
Therapy targeting signal transduction Blood vessels
Therapy targeting LSC microenvironment Endothelial cells

Figure 1 Leukemia stem cells biology and selected therapeutic strategies/agents targeting leukemia stem cell. Leukemia stem cells (LSC) directed therapy
targets cell surface markers expressed on LSC (grey boxes), crucial pathways for maintenance of stemness (orange boxes) and interactions between LSC and the
bone marrow niche (white boxes). Important LSC surface markers are CD33, CD44, CD123, CD47. Essential pathways are NF-κB, PI3K/AKT/mTOR and bcl-2.
LSC mobilization is accomplished with G-CSF and LSC homing to the bone marrow is regulated by the CXCR4–CXCL12 and VCAM-VLA4 axis. VCAM-1: Vascular
cell adhesion protein-1; VLA-4: Very late antigen-4; CXCR4: C-X-C chemokine receptor type 4; SDF: Stromal cell-derived factor 1; PI3K: Phosphatidylinositol-4,5-
bisphosphate 3-kinase; AKT: Protein kinase B; mTOR: Mechanistic target of rapamycin; bcl-2: B-cell lymphoma 2; G-CSF: Granulocyte-colony stimulating factor.

fractions of primary NPM-mutated AML into immuno­
deficient mice, it was shown that approximately one-
-
half of cases had LICs exclusively within the CD34
+
fraction, whereas the CD34 fraction contained normal
[66]
multilineage hematopoietic repopulating cells . Most of
+ -
the remaining cases had LICs in both CD34 and CD34
fractions and when samples were sorted based on
CD34 and CD38 expression, multiple fractions initiated
leukemia in primary and secondary recipients (Table 1).
Heterogeneity within the LSC population
Over the last years several groups have found a wide
variety of other markers that appear to be expressed
higher in LSCs than normal HSCs .
[14]
These include CD123, CD96, CLL-1, TIM3, CD33,
CD13, CD44, CD47 and others
[69-75]
(Table 1). In
essence, these studies suggest that leukemogenic
+ -
activity is not restricted to the CD34 CD38 fraction and
there is heterogeneity among patients in leukemogenic
cell phenotype. Over the last years, there has been
significant advancement in the understanding of the
complexity and heterogeneity of human LSC. Several
important observations have been made along the way
of discovery.
LSC heterogeneity within a patient: First, there is
heterogeneity of the stem cell population within the
same patient as not all LSC have the same self-renewal
[10,76]
capacity . Use of lentiviral gene marking to track the
behavior of individual leukemia initiating cells following
serial transplantation has revealed heterogeneity
in their ability to repopulate secondary and tertiary
recipients and this enabled researchers to classify long
[76,77]
term (LT-LSC) and short term (ST-LSC) LSCs .
LT-LSCs are defined by a long-termed persistence in
xenotransplantion models given an extensive self-
renewal capacity while ST-LSCs have a reduced self-
renewal capacity and only a transient repopulation
capability in xenotransplantation models.
LSC heterogeneity based on the specific xeno­
trans­plantation model used: The LSC phenotype

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 Stahl M et al . AML stem cells

also a recreation of the cytokine environ­ment supporting

Table 1 Markers of leukemia stem cells [14]
stem cell self-renewal and quiescence . This has led to
Cell surface Patient samples used Mouse model Ref. the development of mice models that express human
markers used [13,81]
cytokines like human SCF, GM-SCF, IL3 and TPO .
CD34+CD38- FAB M1, M4, M5 NOD/SCID [15,16]
CD34+CD38+ CN-AML, MLL-ENL NOD/SCID + [18,64,67,68] LSC heterogeneity between patients: It has
IVIG or
become increasingly evident that the LSC phenotype
anti-CD122
CD34-CD38+ AML with NPM1 NOD/SCID β-2 [66] varies between patients based on the specific subtype of
mutation microglobulin leukemia that they suffer from (Table 1). As mentioned
NOD/SCID IL2 above, the majority of AML cells express CD34, however
receptor γ−/− + in AML cells carrying a mutation in NPM1 the CD34
+

IVIG
CD34+CD123+ FAB M1, M2, M4 NOD/SCID [69]
percentage is very low and LSC activity is exclusively
- [66]
CD34+CD38-CD96+ CK-AML, CBFB- Rag2-/- IL2RG-/- [70] restricted to the CD34 population . Furthermore,
MYH11, specific subtypes of AML (in particular less aggressive
PML-RARA, subtypes) are significantly more difficult to be engrafted
AML1-ETO,
as they may have low progenitor cell frequency or are
FAB
M4
particularly sensitive to a specific cytokine or cell type
[14]
CD34+CLL1+ AMLs with FLT3- NOD/SCID [71] missing in the particular xenotransplantation model .
ITD For example, AML samples with a t(8;21) translocation
TIM3+ FAB M1, M2, M4 NOD/Rag1-/- [72] were shown to be difficult to be engrafted and found
IL2RG-/-
to be dependent on signaling though the TPO/mpl
CD34+CD38- CN-AML, NOD/SCID [73] [82,83]
CD33+CD13+ CBF-AML,
pathway . Subsequently, human TPO knock-in mice
MLL-ENL were shown to have improved engraftment for t(8;21)
[84]
AML samples .
FAB: French-American-British classification system; CN: Cytogenetically
normal; CK: Cytogenetically complex; MLL-ENL: Mixed-lineage
leukemia-eleven nineteen leukemia; NPM1: Nucleophosmin 1; CBFB-
Cell of origin of LSCs
MYH11: Core binding factor beta unit-Myosin heavy chain 11; PML- It is important to distinguish the concept of the cell of
[10]
RARA: Promyelocytic leukemia-retinoic acid receptor alpha; AML1-ETO: origin from the CSC . The CSC has stem cell like pro­
Acute myeloid leukemia 1 protein- eight twenty one; FLT3-ITD: Fms- perties and is capable of initiating and sustaining tumor
like tyrosine kinase 3 Internal tandem duplication; NOD/SCID: Non- growth, whereas the cell of origin refers to the normal
obese diabetic/severe combined immunodeficiency; Rag: Recombination
cell in which the initial transforming event occurs. Impor­
activating gene; IL2RG: Interleukin 2 receptor subunit gamma.
tantly, cancer and LSCs do not have to arise from a
normal stem cell, in fact, it is not entirely clear what the
depends on what mouse model is used for functional cell of origin for most LSCs is
[11,12]
. One hypothesis is
assessment of stem cell properties of human AML that LSCs are only able to arise from normal HSCs but
[14]
cells (Table 1). The bone marrow niche in mice differ not from committed progenitor cells
[10,15]
. This theory is
from that of humans in terms of architecture, stromal supported by the observation that LSCs and HSCs share
cells, cytokines, growth factors, adhesion molecules many characteristics like self-renewal capacity controlled
and most importantly the immune cell composition, by genes like Bmi1 and PTEN and quiescence
[35,85,86]
. On
which potentially impairs growth of human HSC or the contrary, transformation might occur in a variety
[78]
LSC in the mouse bone marrow . Normal HSCs and of cell types in the hematopoietic hierarchy, including
LSC are therefore more likely to be detected in more HSCs and committed progenitors
[10,87]
. Experimental
highly immunodeficient mice. As different xenotrans­ evidence in mice shows that LSCs may arise either
plantation mouse models display different levels of through neoplastic changes initiated in normal self-
immunodeficiency they are associated with different renewing HSCs or downstream progenitors cells
[10,11,88]
.
levels of engraftment of normal human HSCs and Some oncogenes including MOZ-TIF, MLL-AF9 and
[6,14]
LSCs . In nude mice T cells are absent whereas in MLL-ENL can induce LSCs regardless of what target
severe combined immunodeficiency mice (SCID mice) cell population they are expressed in
[88-90]
. Other
both B and T cells are inactivated. NOD/SCID mice, oncogenes like BCR-ABL, FLT3-ITD, Hoxa9 and Meis1
which harbor defects in T, B, and macrophage activity, were found to be oncogenic when expressed in HSCs
[14]
support higher levels of human engraftment . NOD/ but not when expressed in progenitor cells
[39,89,91]
.
SCID gamma (NSG) mice have almost no murine However, experimental data in murine studies might be
immune system left as a complete null mutation in the confounded by non-physiologic levels of expression from
gene encoding the interleukin 2-receptor gamma chain exogenous promoters, such as transgenes or retroviral
[79] [11]
blocks NK cell differentiation . Similarly, NK cells can be vectors . This was demonstrated by the recent finding
depleted by treating NOD/SCID mice with anti-CD122 that in an MLL-AF9 knock-in model of the same construct
[80]
antibodies . In creating a supportive bone marrow shown to initiate disease in both HSCs and progenitor
niche for engraftment of human AML cells not only a cells by retroviral expression only initiated leukemia
suppression of the hosts immune system is essential but from HSCs when expressed from the endogenous MLL

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[92]
promoter . In vivo clonality studies in humans suggest
variations in the cells of origin and is was demonstrated
+ -
that in patients with t(8;21) AML primitive CD34 CD90
-
CD38 HSC like cells from leukemic bone marrow give
rise to normally differentiating progenitors, whereas
+ - +
more mature CD34 CD90 CD38 multi-potent progenitor
[93-95]
like cells form exclusively leukemic blast colonies
These observations suggest that the truth about the cell
of origin might be reflected by a combination of both
theories depicted above: Although the initial genetic
mutation might happen in HSCs subsequent events
occur in the committed progenitor pool, giving rise to
[11]
LSCs .
IMPACT OF LSC ON CURRENT
TREATMENT AND PROGNOSIS
Impact on prognosis
The LSC burden of AML patient is suggested to be a
[96-100]
strong biomarker for clinical outcome in AML
ability of cells from AML patients to engraft NOD/SCID
mice and the LSC frequency (simplistically characterized
+ -
as CD34 CD38 frequency) are associated with worse
[99-101]
clinical outcomes . AML patients with greater than
+ -
3.5% of CD34 CD38 AML cells show a median relapse
free survival of 5.6 mo vs 16 mo in those with a lower
+ - [96]
percentage of CD34 CD38 cells . Furthermore,
poor clinical outcome seems to correlate with the
degree to which the LSCs matched normal HSC gene
[98]
expression .
It is noted that it is controversial whether the
sim­plis­tically phenotypically defined LSC frequency
+ -
(charac­terized as CD34 CD38 ) in AML is prognostic
[14]
and correlates with xenograft potential . Also, as
described above, LSCs can be found outside of the
+ -
CD34 CD38 cell fraction. An improved characterization
of subpopulations of LSCs is expected to be associated
with improved prediction of prognosis.
Impact on current therapies
It is thought that LSCs have a significant role in the
relapse of leukemia as induction chemotherapy targets
[102]
the bulk of blast cells but not LSC . Minimal residual
disease (MRD) is an important determinant for relapse
and poor outcomes in AML and it is likely that the MRD
[103-105]
cell population contains LSCs . Thus, in order to
improve outcomes in AML, MRD needs to be reduced
to prevent disease relapse. LSCs seem to be only
[35,106]
minimally affected by traditional chemotherapy
Several reasons for chemotherapy resistance have been
proposed, which are related to the key features of LSCs
discussed above. LSCs are quiescent in the G0 phase of
the cell cycle but chemotherapy is only effective in killing
[36,37]
rapidly cycling cells . LSCs are supported by a stem
cell niche in the bone marrow protecting them from
[65]
the effect of classical chemotherapy . Furthermore,
LSCs express high levels of ATP transporters, which are
involved in extrusion of chemotherapeutic drugs from
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Stahl M et al . AML stem cells
[107-109]
LSCs .
To improve survival in AML, traditional chemotherapy
targeting the blast population needs to be combined
with therapy specifically targeting LSCs to maintain
prolonged remission.
.
FUTURE DIRECTIONS FOR THERAPY
Despite the recent increased interest in LSCs, experi­
mental studies have not been translated into improved
survival outcomes for cancer patients. However, several
new agents targeting LSC specific surface molecules
and pathways as well as the LSC microenvironment
remains under different stages of preclinical and clinical
development (Table 2 and Figure 1). To rationally design
clinical trials testing drugs for efficacy against LSCs, it
is important to appreciate the fundamental differences
[102]
between drug design targeting blast cells and LSCs .
Principles and challenges faced by targeting LSCs will be
discussed first followed by an overview of various new
. The
therapeutic options targeting LSCs.
General principles and challenges faced by targeting
LSCs
Limiting side effects: As LSCs and HSCs have many
similar properties (see above), therapeutic approaches
targeting LSCs also have the potential of causing
severe side effects by eliminating healthy HSCs. To
develop novel therapies with limited side effects,
[102,110]
unique properties of LSCs have to be identified .
While expression of several surface markers is similar
between normal HSCs and LSCs (CD34, CD38, CD71
and HLA-DR), other surface antigens are only displayed
[110]
on LSCs (CD33, CD90, CD117 and CD123) . Apart
from a similar immunophenotype, HSCs and LSCs
share many pathways important for maintaining
features of “stemness” like quiescence and self-renewal
[111]
capacity . Pathways, which are up-regulated in
LSCs compared to normal HSCs, are the ideal target
for therapeutic approaches directed towards LSCs. For
example, the active form of NF-κB and bcl-2, which are
associated with anti-apoptotic activity in cancer cells,
are overexpressed in LSCs compared to normal HSC
and drugs targeting both NF-κB and bcl-2 are in clinical
development
[36,46,112]
.
Using biomarkers for LSC eradication: To assess
the efficacy of investigational therapies targeting
LSCs, precise diagnostic methods are needed to
. assess the quantity of LSCs present in leukemia
patients. Unfortunately, current characterization of LSC
phenotype is not precise enough to permit real-time
[113]
tracking of LSCs in vivo . As discussed above, current
strategies for purification do not yield functionally
homogeneous population: The frequency of LSCs within
+ - 4
the CD34 CD38 fraction in AML ranges from 1 in 10
6
to 1 in 5 × 10 cells and several other populations
[15]
contain LSCs as well . Functional assessment of LSC
frequency with xenotransplantation models offers a
321 October 26, 2016|Volume 8|Issue 10|
 Stahl M et al . AML stem cells

GSK21110183 AKT NCT00881946 Phase [38,155-157]

Table 2 Emerging therapy targeting leukemia stem cells
MK-2206 inhibitors NCT01253447 I-II
Perifosine NCT01231919
Drug Mechanism Selected Phase Ref.
NCT00301938
clinical trials
Sirolimus, mTOR NCT01184898 Phase [38,158]
Therapy targeting cell surface markers everolimus, inhibitors NCT01611116 I-II
GO Anti-CD33 NCT00882102 Phase [124,126,­ temsirolimus NCT01074086
monoclonal NCT01869803 I-III 130,132,133] NCT01074086
antibody NCT00968071 NCT01154439
conjugated NCT01409161 NCT00775593
with NCT00766116 NCT02583893
calicheamicin, NCT02724163 NCT01869114
a potent NCT00658814 NCT01822015
antitumor NCT02473146 Oblimersen bcl-2 antisense NCT00085124 Phase [46,159,160]
anthracycline NCT00895934 (Genasense, oligodeoxy­ NCT00039117 I-III
antibiotic NCT00006265 G3139) nucleotide NCT00017589
NCT00860639 Obatoclax Small NCT00438178 Phase [161-163]
NCT00927498 Mesylate molecule bcl-2 NCT00684918 I-II
NCT00085709 (GX15-070MS) inhibitor NCT00684918
NCT00195000 Therapy targeting the LSC microenvironment
NCT00893399 G-CSF Mobilization NCT00820976 Phase [165-168]
NCT00017589 of LSC from NCT00602225 I-III
Hu5F9-G4 Anti-CD47 NCT02678338 Phase [74,141] the protective NCT00199147
monoclonal I bone marrow NCT01723657
antibody niche - > NCT01101880
CSL360 Anti-CD123 NCT00401739 Phase [69,134] increased NCT00943943
monoclonal I susceptibility NCT00906945
antibody to traditional
DT388IL3/ Anti-CD123 NCT02113982 Phase [69,134,136] chemotherapy
SL-401 recombinant NCT00397579 I-II Plerixafor CXCR4 NCT00943943 Phase [61,135,178]
immunotoxin (AMD3100) inhibitor NCT00906945 I-II
created by Decreased NCT01236144
the fusion of homing to the NCT00512252
diphtheria bone marrow NCT01319864
toxin with NCT01352650
a ligand NCT02416908
targeting the AMD3465 CXCR4 N/A N/A [61,135,169,179]
IL-3 receptor inhibitor
MGD006/ Anti-CD3 and NCT02152956 Phase [137] Decreased
S80880 CD123 DART I homing to the
H90 Anti-CD44 N/A N/A [75,139] bone marrow
monoclonal BMS-936564 Anti-CXCR4 NCT01120457 Phase [172]
antibody antibody I
A3D8 anti-CD44 N/A N/A [139] Decreased
monoclonal homing to the
antibody bone
Therapy targeting LSC-specific molecular pathways marrow
Bortezomib Proteasome NCT00789256 Phase [36,143-147,­ Natalizumab Anti-VLA4 N/A N/A [174]
inhibitor NCT00382954 I-III 175-177] antibody
inhibits the NCT01127009 Decreased
degradation NCT00666588 homing to the
of the IkBa NCT00703300 bone marrow
creating an NCT01534260
anti-NF-kB NCT00383474 GO: Gemtuzumab ozogamicin; DART: Dual-affinity retargeting molecule;
effect N/A: Not available; LSC: Leukemia stem cell; IkBa: Inhibitor of kappa
Parthenolide Inhibitor of N/A N/A [149] B alpha; CXCR4: C-X-C chemokine receptor type 4; mTOR: Mechanistic
NF-kB target of rapamycin; G-CSF: Granulocyte-colony stimulating factor.
Celastrol Inhibitor of N/A N/A [150]
Hsp90 and by
extension NF- more robust method to evaluate eradication of LSCs but
kB [102]
might not be feasible in large clinical trials . Similarly,
4-hydroxy- Product N/A N/A [151,152]
2-nonenal of lipid methods for detecting MRD might guide decisions by
peroxidation, detecting patients who do require additional therapy
inhibiting the to prevent relapse. However, detecting MRD does not
proteasome distinguish persistent LSCs, which may cause relapse,
and NF-kB
from residual blasts and normal HSCs that do not have
function
BKM120 PI3K NCT01396499 Phase [38,153,154] tumor-initiating activity. Distinguishing residual LSCs
CAL-101 inhibitors NCT01833169 I-II from residual blasts might be accomplished by gene
NCT00710528 expression analysis showing reactivation of self-renewal

WJSC|www.wjgnet.com 322 October 26, 2016|Volume 8|Issue 10|


[88,114]
genes in LSCs but not in blast cells . In preclinical
development, the recently published Connectivity Map
could be investigated for agents that attenuate a stem
[115,116]
cell gene signature or induce a differentiated state . disease-free survival and OS- despite no differences in
Timing of LSC targeted therapy: Therapy targeting
LSCs is effective in eradicating a small amount of
leukemia initiating cells but not the bulk of blasts
[102]
cells in the blood and bone marrow . By combining
drugs eradicating LSCs with standard chemotherapy
targeting the bulk of the disease, both the aggressive
proliferating process as well as the root of the leukemia
[117]
can be targeted . An example serves the successful
combination of the anti-CD33 immunoconjugate anti­
body gemtuzumab ozogamicin (GO) with standard
[118]
chemotherapy . This is associated with challenges
in a meaningful design of clinical trials in terms of the
correct timing of these therapies. LSC targeting therapy
can either be given after reduction of the bulk population
with standard chemotherapy as remission therapy
or concomitant with chemotherapy as an induction
[102]
regimen . Upfront combination would allow assessing
for additive and/or synergistic properties between drugs
and would allow targeting of LSCs early on in the disease
[102]
process, which might improve outcomes . On the
other hand, LSC targeted therapy might be particular
valuable as post-consolidation therapy as no current
post-consolidation intervention has led to improved OS
[102,119,120]
for patients with AML . LSC targeting therapies
have the potential to fill the gap as they eradicate the
cells responsible for relapses of AML.
Assessing clinical endpoints: Classical response
criteria like CR and hematologic improvement might
not be the best parameters to assess the efficacy of
therapeutic approaches targeting LSCs as these drugs
do not eradicate the bulk of blast cells but rather
[102]
eliminate the rare population of LSCs . Progression-
free survival (PFS), event free survival and overall
survival (OS) may be a more relevant endpoint for
assessing the effectiveness of LSC elimination than
tumor response as they better account for whether
[113]
the root of the leukemia has been eliminated . chemotherapeutic agents (chemo-immune conjugates)
Importantly, while LSC frequency was found to be
prognostic for survival, response rates did not correlate
[96]
with LSC burden . Subsequently, drugs targeting LSCs
may show little activity if tested in traditional phase
I/II trials as a proper assessment of endpoints relevant
for LSCs, like PFS and OS, is generally only feasible in
a phase III trial with a larger numbers of patients and
[113,121]
long-term follow-up . others .
One example for the importance of assessing
relevant endpoints for LSC targeting therapy, is incon­
sistency of clinical trials evaluating the efficacy of
[102]
GO . Single agent studies of GO showed overall
response rates only approaching 30% at best and
GO was voluntarily withdrawn from the United States
market in 2010 after a study showed no improvements
in outcomes when used in combination therapy as well
WJSC|www.wjgnet.com
Stahl M et al . AML stem cells
[122-124]
as increased fatal toxicity . In contrast, other large
clinical trials showed improvement in outcomes more
relevant for therapies targeting LSC- event-free survival,
[125-128]
disease response rates .
Targeting LSC surface molecules
Anti-CD33 antibodies: CD33 is found on LSCs alth­
ough it is not a consistent feature of all LSCs studi­
ed
[73,118,129]
. As discussed above, there have been con­
flicting reports surrounding the efficacy and safety of
GO and currently GO is not available on the market in
[130]
the United States or Europe . Apart from the different
endpoints studied, there are additional explanations
for the discrepancies observed: First, the dose of
daunorubicin as the combination partner of GO did vary
between trials, although it is known that treatment with
daunoru­bicin-based schedules of 90 mg/m for 3 d is
2
more effective than similar schedules with daunorubicin
at 45 mg/m
2[131]
. In the SWOG trial, which questioned
the efficacy of GO, single bolus combined with dau­
2
norubi­cin at 45 mg/m was studied against a control
group with daunorubicin at 60 mg/m
2[124]
. However, the
best effect of GO was seen when higher dose of GO (3
2
d at 3 mg/m for 2 cycles) was added to a daunorubicin
2
regimen of 60 mg/m in both comparator groups .
[126]
Furthermore, GO seems to be quite active in acute
promyelocytic leukemia (APL) as APL cells express high
levels of CD33
[132,133]
. These results have prompted calls
to reconsider the approval status of GO .
[130]
Anti-IL-3 receptor (CD123) antibodies: The inter­
leukin-3 receptor alpha chain (IL-3Rα or CD123) is
+ -
strongly expressed in CD34 /CD38 LSCs and can be
targeted with monoclonal antibodies
[69,134]
. The blockage
of CD123 has pleiotropic anti-leukemic effects including
inhibition of LSC homing to the bone marrow, activation
of innate immunity and inhibition of intracellular signa­
ling events
[135]
. Several different agents targeting
CD123 are currently evaluated in clinical trials: CD123
targeting antibodies can either be naked antibodies
or be conjugated to toxins (e.g., diphteriod toxin) or
or be the backbone of a bi-specific T cell engager (BITE,
[134,136,137]
e.g., CD3-CD123) (Table 2).
Anti-CD44 antibodies: CD44 regulates interaction
between LSCs and the bone marrow niche by controlling
cell-cell adhesion and cell-matrix interaction through
binding to hyaluronic acid, osteopontin, collagens and
[138]
Inhibition of CD44 with monoclonal antibodies was
shown to reduce the numbers of LSCs in NOD/SCID
mice and to increase the survival of the primary reci­
pient mice as well prevent engraftment into the secon­
[75,139]
dary receipt mice (Table 2).
Anti-CD47 antibodies: CD47 is overexpressed on
LSCs and high expression of CD47 is associated with
323 October 26, 2016|Volume 8|Issue 10|
 Stahl M et al . AML stem cells
[74]
worse outcomes . By interaction with the extracellular
region of signal-regulatory protein alpha (SIRPα)
on phagocytic cells, LSCs deliver a “do not eat me”
[140]
message to these phagocytic cells . Antibodies
blocking the interaction between CD47 and SIRPα
promote LSC phagocytosis and are in development
[74,141]
(Table 2) . a higher rate of disease-free survival than patients
Targeting LSC-specific molecular pathways
NF-κB signaling pathway: Bortezomib is able to
suppress the NF-κB signaling pathway by inhibiting the
destruction of IκB, a cellular inhibitory protein of NFκB,
[142]
by the ubiquitin-proteasome pathway . Several clinical
trials are examining the efficacy of Bortezomib targeting
AML LSCs (Table 2): Two clinical trials combining
Bortezomib with Cytarabine and Anthracyclines resulted
in CR rates of 61% and 65%
[143,144]
, whereas other trials
that co-administrated Bortezomib with other drugs did
not show encouraging CR rates
[145-147]
. Several other
inhibitors of NF-κB signaling are in different phases of
development (Table 2)
[148-152]
. previous treatment. Several clinical trials are ongoing
PI3K/AKT/mTOR pathway: The PI3K/AKT/mTOR
pathway is of utmost importance in regulating cellular
growth, survival, and metabolism and is frequently
[38]
dysregulated in cancers and AML . A multitude of
PI3K inhibitors
[153,154]
, AKT inhibitors
[155-157]
and mTOR
inhibitors
[158]
is currently investigated for their efficacy
targeting LSCs in clinical trials (Table 2).
Bcl-2 pathway: LSCs, similar to other tumor cells,
are able to avoid apoptosis due to overexpression of
[46]
bcl-2 . Currently, bcl-2 inhibition is investigated in
clinical trials in form of the bcl-2 antisense oligodeo­
xynucleotide oblimersen
[159,160]
and the small molecule
inhibitor of bcl-2 obatoclax
[161-163]
(Table 2).
Targeting the LSC microenvironment
Approaches targeting the interactions of LSCs with the
bone marrow niche focus on breaking the dormancy
of LSCs in the bone marrow in order to make them
sensitive to traditional chemotherapy
[62,164]
. examined the efficacy of the CXCR4 inhibitor plerixafor
LSC mobilization: LSC mobilization from the marrow
niche can be achieved by nonspecific stimulators like
[62]
G-CSF, Interferon-α and Arsenic trioxide . Using the
NOD/SCID/IL2rgamma (null) mouse model, Saito
[165]
et al showed that quiescent human AML LSCs, at
first resistant to cytarabine, start proliferating and
become susceptible to cytarabine once exposed to
G-CSF. Combining chemotherapy with G-CSF leads to
significantly increased survival of secondary recipients
after transplantation of leukemia cells compared with
chemotherapy alone. Furthermore, they showed that
treatment with G-CSF before cytarabine did not increase
apoptosis of normal HSCs making this approach a
particular attractive option for targeting LSCs but at
the same time avoiding side effects from depletion of
HSCs. The data from clinic trials using G-CSF priming
WJSC|www.wjgnet.com
in combination with chemotherapy are conflicting.
[166]
Löwenberg et al randomized 640 newly diagnosed
AML patients to receive cytarabine plus idarubicin with
G-CSF (321 patients) or without G-CSF (319 patients)
for the first cycle of induction of chemotherapy. Patients
in CR after induction chemotherapy plus G-CSF had
who did not receive G-CSF (42% vs 33% at four
years, P = 0.02), owing to a reduced probability of
relapse (relative risk, 0.77; P = 0.04). Other studies
did not show a benefit of adding G-CSF to traditional
[167,168]
chemotherapy regimens . These different res­
ponses to G-CSF might be explained by differences in
[63]
the group of patients included in these trials . In the
[166]
trial by Löwenberg et al patients with standard-risk
AML benefited from G-CSF therapy whereas G-CSF
did not improve the outcome in the subgroup with an
unfavorable prognosis. In the trials without improvement
with G-CSF, patients had a more unfavorable prognosis
based on age, cytogenetic abnormalities or response to
to investigate the efficacy of G-CSF in combination of
chemotherapy in different risk groups of AML (Table 2).
Inhibition of homing: LSC dormancy can be targeted
by specifically interrupting the CXCR4-CXCL12 and
VCAM-VLA4 axis as well as inhibiting CD44 and CD123
on LSCs to prevent homing of LSCs to the bone
marrow.
CXCR4-CXCL12 axis: SDF-1 was shown to promote
survival of AML cells, whereas addition of neutralizing
CXCR4 antibodies, SDF-1 antibodies, or AMD3100
[169]
significantly decreased their survival . Furthermore,
pretreatment of primary human AML cells with
neutralizing CXCR4 antibodies blocked their homing into
null
the BM and spleen of transplanted NOD/SCID/B2m
[169]
mice . Additionally, CXCR4 inhibition with AMD3465
was shown to increase the sensitivity of FLT3-mutated
leukemic cells to the apoptogenic effects of the FLT3
[170]
inhibitor sorafenib . Recently a phase 1/2 study
in combination with mitoxantrone, etoposide, and
cytarabine in 52 patients with relapsed or refractory
[171]
AML . Overall CR was found to be 46% and corre­
lative studies demonstrated a 2-fold mobilization in
leukemic blasts into the peripheral circulation without
evidence of symptomatic hyperleukocytosis or delayed
count recovery. BMS-936564, a fully human IgG4
monoclonal antibody against CXCR4, exhibits antitumor
activity in cytarabine-resistant mouse xenograft models
of AML and is currently tested in a phase I clinic trial
[172]
(Table 2) .
VCAM-VLA4 axis: Integrin alpha4beta1 (VLA4)
mediates adhesion of LSCs to stromal cells and extra­
cellular matrix in the marrow niche and can be blocked
[59,69,173]
by the monoclonal antibody Natalizumab .
AML cells were shown to de-adhere from a layer of
324 October 26, 2016|Volume 8|Issue 10|

immobilized human VCAM1 expressing human stromal
cells when exposed to Natalizumab and NSG mice
transplanted with human AML cells survived significantly
longer when they received intraperitoneal Natalizumab
[174]
injections .
CONCLUSION
AML remains one of the most difficult malignancies
to treat. Despite significant advancements in the
understanding of disease biology, this has not been
translated yet into new treatment modalities improving
outcomes. The relapse of AML is frequent and is
responsible for the inability to cure AML. LSCs are
understood to be the root of relapse and their presence
has been found to be prognostic for the disease course.
Unfortunately, LSCs are not easy to target as they are
quiescent, able to self-renew and well protected by
a supportive bone marrow niche. Furthermore, their
inconsistent phenotype and similarity to normal HSCs
hamper specific drug development. Nevertheless, a
multitude of potential targets have been identified
and are currently tested in different phases of clinical
and preclinical development. Successful eradication of
LSCs will require combination of different strategies
including targeting LSC specific surface molecules
and pathways as well as interactions of LSCs with the
microenvironment. Furthermore, clinical trials have
to be designed in a way that they are able to detect
a specific effect of LSCs, which is easy to miss in a
traditional trial design. Overall, targeting LSCs has the
promise to not only effectively reduce disease burden
but to eradicate the root of leukemia itself.
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P- Reviewer: Kita K, Li ZJ, Ramírez M, Shao R S- Editor: Ji FF
L- Editor: A E- Editor: Wu HL
331 October 26, 2016|Volume 8|Issue 10|
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